Cryptosporidium and CryDtosporidiosis
Cryptosporidium is a genus of protozoan parasite commonly found in the gastrointestinal tract of vertebrates. There are eight named species of Cryptosporidium, C. parvum is infectious for 79 species of mammals, including humans, causing acute gastroenteritis. Unlike most parasites, C. parvum lacks host specificity among mammals and is able to cross- infect multiple host species.
Cryptosporidiosium is transmitted as an oocyst via the fecal-oral route. Contaminated water, close human-to-human contact and contact with the excrement of infected livestock, zoo animals or domestic animals can lead to transmission of the parasite. When outside a host, the exogenous phase, Cryptosporidium exists as a sporulated oocyst. The oocyst consists of four sporozoites within a tough, two layered wall. The oocyst wall has defined inner and outer layers and a suture at one end. During excystation, the suture dissolves providing an opening through which the sporozoites leave the oocyst. When ingested by a suitable host, excysted sporozoites, the living matter, parasitize the cells of the gastrointestinal or respiratory tract. After reproduction, resporulation into oocysts occurs. Oocysts in the gastrointestinal tract are excreted with the fecal matter while those in the respiratory tract exit the body in respiratory and nasal secretions.
Cases of infection by Cryptosporidium are commonly encountered in both developed and underdeveloped countries. The slightly higher prevalence of Cryptosporidiosis in the lesser developed countries can be attributed to poor sanitation, malnutrition, contaminated drinking water and close contact with infected persons and animals.
The most common clinical sign associated with Cryptosporidiosis is diarrhea, which can be severe and result in weight loss and dehydration. Other common clinical symptoms include abdominal cramps, fever, nausea, vomiting, headache, fatigue, myalgia and inappetence. Infections with Cryptosporidium are generally in the small intestine, but have also occurred in the lungs, esophagus, stomach, and other organs. The clinical symptoms associated with the parasitic infection depend on the affected organ.
The range of symptoms and the severity of the illness can vary greatly from one individual to another and can become life threatening. The symptoms of acute enteritis generally last one to two weeks in individuals who are otherwise immunologically healthy. Cryptosporidiosis represents a heightened threat in AIDS patients, malnourished persons, individuals with inherited immune deficiencies, and person receiving immunosuppressive drugs.
All infections with Cryptosporidium are initiated by ingestion or inhalation of the oocyst. Because the parasite is transmitted in the form of an oocyst, oocysts have evolved to survive in harsh environmental conditions and are unusually resistant to natural stresses and chemical disinfectants. In addition, the presence of an exogenous oocyst encapsulating the protozoan parasite makes the parasite much more resistant to conventional water treatment processes. Measures to prevent or limit the spread of infection concentrate on eliminating or reducing infectious oocysts in the environment. For humans, disinfection procedures are sought to minimize person-to-person transmission and to deal effectively with contamination of water supplies.
The fairly recent occurrence of large water-borne outbreaks has focused attention on the importance of understanding their transmission through the environment. Surface waters may be polluted naturally by infected animal excrement. Many waste disposal practices may lead to contaminated water courses and streams. Fecal contamination of waterways has recently led to massive outbreaks of C. parvum infection. Water polluted by these practices may then lead to the contamination of drinking water supplies or of food crops during irrigation.
Chemical Composition and Decomposition of Cryptosporidium
The protein, carbohydrate, and lipid composition of C. parvum is diverse and complex. Many of the components are antigenic and therefore function as immune response targets. Glycoproteins ranging from &lt;14 to 7200 kDA from disrupted oocytes, purified oocyst shells and purified sporozoites have been identified by SDS-PAGE gel electrophoresis. Many of these oocyte-derived proteins are glycosylated. Specific carbohydrate moieties have been identified. It has been determined that sporozoite glycoproteins with terminal N-acetyl-D-glycosamine residues may function in attachment of the parasite or somehow assist in invasion. These carbohydrates are expressed on the oocyte surface and are useful in immunological detection methods. sporozoites of Cryptosporidium can spontaneously excyst through a suture at one pole of the oocyst when warmed to about 37.degree. for approximately 90 minutes. This renders mechanical methods for oocyst wall disruption unnecessary to accomplish.
Pretreatment of C. parvum oocytes with sodium hypochlorite (a "bleach") results in separation of the inner and outer oocyst walls. That is, while it is not necessary to pretreat oocysts within a reducing agent when excysting C. parvum, a slight increase in the rapidity of excystation occurs when bleach treated oocysts are incubated in PBS containing 0.01M cysteine HCL during the excystation process. Oocysts that have not been pretreated with bleach excyst somewhat when they are warmed to approximately 37.degree. C. The use of trypsin and bile salts, or bile salts alone, can increase or speed excystation of unbleached oocysts.
Prior Art Assay Methods for Crytospyridium
Immunological techniques have been used to detect C. parvum in environmental specimens. The availability of monoclonal antibodies for specific antigens of Cryptosporidium facilitated development of these methods.
Immunofluorescence assays (IFA) are the most common assays used to detect Cryptosporidium oocytes in specimens and to detect the presence of a specific antibody. These methods employ fluorescent dyes which are combined with antibodies to make them fluoresce when exposed to ultraviolet light. In a typical IFA assay, water is filtered through a polypropylene cartridge filter or a flat, membrane filter. Both filters yield filtrates that are then subjected to purification before analysis by microscopy. The filtrate is removed from the filter and then centrifuged. Extraneous debris is removed by flotation over a sucrose solution. The supernatant is labeled with a fluorescein conjugated antibody against Cryptosporidium and examined by epifluorescence microscopy.
Some commercial immunofluorescent assays and reagents used to detect Cryptosporidial oocytes include: (1) HydroFluor Combo, an immunofluorescent assay system based on an oocyst-specific monoclonal antibody (IgM, OW3) (2) Detect IF Cryptosporidium, an immunofluorescent assay system based on an oocyte-specific monoclonal antibody (IgM, C1), and (3) Crypto IF Kit, an immunofluorescent assay system based on an oocyst-specific monoclonal antibody.
The disadvantages of immunofluorescence assays include their low recovery efficiency, long processing times, the need for highly trained analysts, high cost, the inability to discriminate viable or virulent strains and cross-reactivity of the probes with similar size and shaped algae. In addition, IFA detection often involves the time consuming and skill intensive step of looking at water sludge under a microscope for oocysts that have been labeled with a fluorescent antibody. It is also often difficult to distinguish oocysts from debris bound non-specifically by the antibodies. The procedure is expensive and often takes days to complete.
Enzyme-linked immunosorbent assays (ELISA) using oocyte-reactive monoclonal antibodies is also used to detect Cryptosporidium in contaminated water samples. Two basic ELISA tests have been used in the past for detecting Cryptosporidium antigen in samples: (1) the double antibody sandwich technique for the detection of antigens, and (2) the enzyme-linked indirect immunosorbent assay for the detection of antibodies.
In the double antibody sandwich method, antiserum is adsorbed to a well. Test antigen is added and, if complementary, binds to the antibody. An enzyme-linked antibody specific for the test antigen then binds to the antigen, forming a sandwich. The enzyme's substrate is then added, and the reaction produces a visible color change. In the indirect immunosorbent assay, an antigen is adsorbed to a well. Test antiserum is then added, with complementary antibody binding to the antigen. Enzyme-linked anti-human gamma globulin is then added. It binds to the bound antibody. The enzyme's substrate is then added, producing a visible color change. A difficulty encountered in enzyme-based assays is the deactivation of the enzyme by components of the assay mixture. A further difficulty is encountered in the wash step where strong forces overcome the antibody-antigen interaction. This leads to loss of assay precision.
Detection assays based upon polymerase chain reactions (PCR) have also been used to detect oocysts in clinical or environmental samples. Several DNA and RNA regions of C. parvum have been sequenced and have been reported to be assay targets for parasite detection.
Flow cytometry is another method used to detect parasitic contamination of water samples. Flow cytometry techniques can quantify whole oocysts but involves much preparation, and time and require extremely expensive equipment.
Numerous problems are associated with prior art methods of detecting Cryptosporidium in water and environmental samples. In addition to those mentioned and the general lack of precise, recitable assays, prior art techniques generally require that samples be transferred to a laboratory or to another remote location for the conduct of the assay. Prior art techniques lack the requisite reliability, speed and sensitivity to accurately detect Cryptosporidium in contaminated water samples.
The detection of infectious C. parvum oocysts in water and other environmental samples is essential to detecting and treating contaminated water supplies. It is crucial, therefore, that specific, rapid and highly sensitive assays be developed to detect the presence of the parasite accurately and reliably. The known methods of enzyme immunoassays and immunofluorescence do not fulfill these requirements. The source, viability and pathogenicity of oocysts found in water or other environmental samples cannot be reliably determined using prior art methods. There is a need for routine epidemiological surveillance and environmental monitoring that can be conducted on site to provide early detection of the parasite.